Cartilage-derived morphogenetic proteins

ABSTRACT

Nucleotide and amino acid sequences of cartilage-derived morphogenetic proteins (CDMP-1 and CDMP-2) from human and bovine cartilage extracts. These proteins exhibit chondrogenic activity and can be used to repair cartilage defects in a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/379,830, filed Mar. 3, 2003, which is a continuation of U.S.application Ser. No. 09/730,772, filed Nov. 30, 2000, now abandoned,which is a continuation of U.S. application Ser. No. 08/836,081, filedJul. 28, 1997, now abandoned, which represents the U.S. national phaseof International Application No. PCT/US94/12814, filed Nov. 7, 1994,designating the United States of America and published in English on May17, 1996 as WO 96/14335.

FIELD OF THE INVENTION

The present invention relates generally to the field of cartilage andbone development. More specifically, the invention relates tocartilage-derived morphogenetic proteins that stimulate development andrepair of cartilage in vivo.

BACKGROUND OF THE INVENTION

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamilythat can induce endochondral bone formation in adult animals. Thissuperfamily includes a large group of structurally related signalingproteins that are secreted as dimers and then cleaved to result inbiologically active carboxy terminal domains of the proteins. Thesebioactive proteins are characterized by 7 highly conserved cysteineresidues. Interestingly, these proteins have different roles at variousstages of embryogenesis and in adult animals. Recombinant BMPs are nowavailable and have been shown to induce endochondral bone formation whenassayed in vivo.

Indeed, the initial discovery of the BMPs was facilitated by such invivo assays for cartilage and bone development. These assays were basedon the observation that bone development could be initiated bysubcutaneous or intramuscular implantation of compositions comprising anextract of demineralized bone and residual bone powder. The novelproteins identified in the extracts were termed “bone morphogeneticproteins.” These proteins were subsequently classified as members of theTGF-β superfamily by virtue of amino acid sequence relatedness.Screening of genomic and cDNA libraries led to the isolation ofpolynucleotides encoding BMP-2, -3, -4, -5, -6 and -7.

One deficiency of the bone induction assay regards its inability todistinguish the physiological roles of different BMP family members. Thecartilage and bone inducing activity of the BMPs is remarkable becausethe normal stages of endochondral bone formation that occur duringontogeny are recapitulated in the adult animal. These stages includemesenchymal condensation, cartilage and bone and bone marrow formationand eventual mineralization to produce mature bone.

Several observations suggest that BMPs have wide-ranging extraskeletalroles in development. First, localization studies in both human andmouse tissues have demonstrated high levels of mRNA expression andprotein synthesis for various BMPs in kidney (BMPs-3, -4, -7), lung(BMPs-3, -4, -5, -6), small intestine (BMPs-3, -4, -7), heart (BMPs-2,-4, -6, -7), limb bud (BMPs-2, -4, -5, -7) and teeth (BMPs-3, -4, -7).Second, several members of the family, including BMP-4 and -7, are keymolecules in epithelial-mesenchymal interactions, for instance duringodontogenesis. Third, BMP-2 and BMP-4 are involved in the signalingpathway that controls patterning in the developing chick limb and BMP-4is a ventralizing factor in early Xenopus development. Fourth,Drosophila homologs of the BMPs, the decapentaplegic (dpp) and 60 A geneproducts, have the capacity to induce bone in mammals whereas humanBMP-4 confers normal embryonic dorso-ventral patterning in Drosophilatransformants defective in dpp expression. Thus, the BMPs are nowappreciated as pleiotropic cytokines.

Interestingly, none of the known BMPs are strongly expressed in thechondroblasts and chondrocytes of the cartilage core of developing longbones. The hypertrophic chondrocytes, where both Vgr-1 (BMP-6, (Lyons etal., 1990 Development 109:833) and OP-1 (BMP-7)(Vukicevic et al., 1994Biochem. Biophys. Res. Commun. 198:693) have been found are exceptionsin this regard.

SUMMARY OF THE INVENTION

One aspect of the present invention is a purified cartilage extract thatstimulates local cartilage formation when combined with a matrix andimplanted into a mammal. This extract can conveniently be produced by amethod which includes the steps of: obtaining cartilage tissue;homogenizing the cartilage tissue in the presence of chaotropic agentsunder conditions that permit separation of proteins from proteoglycans;separating the proteins from the proteoglycans and then obtaining theproteins. The step for separating the proteins from the proteoglycanscan be carried out using a sepharose column. The extract can also beobtained by additionally including the steps of separating the proteinson a molecular sieve and then collecting the proteins having molecularweights in the 30 kDa to 60 kDa size range. Articular cartilage orepiphyseal cartilage can be used in the preparation of this purifiedextract.

A second aspect of the present invention is a method of preparing apartially purified articular cartilage extract having chondrogenicactivity. This method includes the steps of first obtaining cartilagetissue; homogenizing the cartilage tissue in the presence of chaotropicagents under conditions that permit separation of proteins fromproteoglycans; separating the proteins from the proteoglycans andfinally obtaining the proteins. The separation of proteins andproteoglycans can be accomplished using a sepharose column. Inparticular, the step for separating proteins from proteoglycans caninclude isolating the proteins that bind heparin Sepharose in thepresence of 0.15 M NaCl but not in the presence of 1 M NaCl. Anadditional step in the purification procedure can include separating theproteins on a molecular sieve and then obtaining the proteins havingmolecular weights between 30 kDa and 60 kDa.

A third aspect of the present invention is an isolated DNA molecule thatencodes a protein having chondrogenic activity in vivo but substantiallyno osteogenic activity in vivo. More particularly, this aspect of theinvention regards a molecule having a nucleotide sequence that canhybridize to a polynucleotide which has the nucleotide sequence SEQ IDNO: 11 or SEQ ID NO: 12 at 55° C. with 0.4×SSC and 0.1% SDS. Theproteins encoded by such DNA molecules can have the amino acid sequencesof SEQ ID NO: 13 or SEQ ID NO: 14.

A forth aspect of the present invention is a recombinant protein havingchondrogenic activity in vivo but substantially no osteogenic activityin vivo. This protein can have the amino acid sequence of SEQ ID NO: 13or SEQ ID NO: 14.

A fifth aspect of the present invention is a method of stimulatingcartilage formation in a mammal. This method includes the steps:supplying cartilage-derived morphogenetic proteins having in vivochondrogenic activity; mixing the partially purified proteins with amatrix to produce a product that facilitates administration of thepartially purified proteins and implanting this mixture into the body ofmammal to stimulate cartilage formation at the site of implantation. Thepartially purified cartilage-derived morphogenetic proteins can beobtained from either articular cartilage or epiphyseal cartilage. Thematrix can also include non-cellular material. Viable chondroblast orchondrocytes can also be included in the mixture prior to implantation.The mixture can be implanted either subcutaneously or intramuscularly.

A sixth aspect of the present invention is a composition that can beadministered to a mammal for the purpose of stimulating chondrogenicactivity at the site of administration without substantially stimulatingosteogenic activity. This composition comprises at least onecartilage-derived morphogenetic protein and a matrix. Thecartilage-derived morphogenetic protein can be derived from an extractof either articular cartilage or epiphyseal cartilage. In anotherembodiment, the cartilage-derived morphogenetic protein is a recombinantprotein. This recombinant protein can have the amino acid sequence ofeither SEQ ID NO: 13 or SEQ ID NO: 14. The matrix used to create thecomposition can be either fibrin glue, freeze-dried cartilage, collagensor the guanidinium-insoluble collagenous residue of demineralized bone.Alternatively the matrix can be a non-resorbable matrix such astetracalcium phosphate or hydroxyapatite.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the nucleotide and predicted amino acid sequence encodedby the full length human CDMP-1 cDNA. Nucleotide is SEQ ID NO: 11. AminoAcid is SEQ ID NO: 13.

FIG. 2 presents the nucleotide and predicted amino acid sequence encodedby the bovine CDMP-2. Nucleotide is SEQ ID NO: 12. Amino Acid is SEQ IDNO: 14.

FIG. 3 presents the genetic maps of chromosome 2 showing thelocalization of CDMP-1. The map on the right is based on the data fromtwo separate crosses.

FIG. 4 shows an alignment of segments from predicted CDMP amino acidsequences in standard one letter code.

DETAILED DESCRIPTION OF THE INVENTION

We discovered that partially purified extracts of newborn calf articularcartilage contained an activity that induced cartilage formation whenimplanted subcutaneously in rats. This biological activity wasreminiscent of that which characterized the BMPs. Degenerateoligonucleotide primer sets derived from the highly conservedcarboxy-terminal region of the BMP family were employed in reversetranscription-polymerase chain reactions (RT-PCR) using poly(A)⁺ RNAfrom articular cartilage as a template. These procedures allowed us todetermine which BMPs were expressed in chondrocytes.

Two novel members of the TGF-β superfamily were identified anddesignated Cartilage-Derived Morphogenetic Protein-1 (CDMP-1), and -2(CDMP-2). The C-terminal TGF-β domains of these proteins were 82%identical, thus defining a novel subfamily most closely related toBMP-5, BMP-6 and osteogenic protein-1. Northern analyses showed thatpostnatally both genes were predominantly expressed in cartilaginoustissues. In situ hybridization and immunostaining of sections from humanembryos showed that CDMP-1 was predominantly found at the stage ofprecartilaginous mesenchymal condesation and throughout thecartilaginous cores of the developing long bones. CDMP-2 expression wasrestricted to the hypertrophic chondrocytes of ossifying long bonecenters. Neither gene was detectable in the axial skeleton during humanembryonic development. The cartilage-specific localization pattern ofthese novel TGF-β superfamily members, which contrasts with the moreubiquitous presence of other BMP family members, suggested a role forthese proteins in chondrocyte differentiation and growth of long bones.

The discovery of a novel subfamily of cartilage derived morphogeneticproteins suggested the existence of morphogens that primarily finctionedin the induction and maintenance (i.e., balancing cartilage and boneformation at articular surfaces) of cartilaginous and bony tissues. Thissubfamily may also include key molecules that govern bone marrowdifferentiation.

The cartilage-derived morphogenetic proteins contained in the cartilageextract of the present invention, and the recombinant CDMP-1 and CDMP-2proteins described herein are contemplated for use in the therapeuticinduction and maintenance of cartilage. For example, local injection ofCDMPs as soluble agents is contemplated for the treatment of subglotticstenosis, tracheomalacia, chondromalacia patellae and osteoarthriticdisease. Other contemplated utilities include healing of joint surfacelesions (e.g. temporomandibular joint lesions or lesions inducedposttraumatically or by osteochondritis) using biological deliverysystems such as fibrin glue, freeze-dried cartilage grafts, andcollagens mixed with CDMPs and locally applied to fill the lesion. Suchmixtures can also be enriched with viable cartilage progenitor cells,chondroblasts or chondrocytes. We also contemplate repair orreconstruction of cartilaginous tissues using resorbable ornon-resorbable matrices (tetracalcium phosphate, hydroxyapatite) orbiodegradable polymers (PLG, polylactic acid/polyglycolic acid) coatedor mixed with CDMPs. Such compositions may be used in maxillofacial andorthopedic reconstructive surgery. Finally, the CDMPs disclosed hereinhave utility as growth factors for cells of the chondrocyte lineage invitro. Cells expanded ex vivo can be implanted into an individual at asite where chondrogenesis is desired.

We also anticipate the polynucleotides disclosed herein will also haveutility as diagnostic reagents for detecting genetic abnormalitiesassociated genes encoding CDMPs. Diagnostic testing could be performedprenatally using material obtained during amniocentesis. Any of severalgenetic screening procedures could be adapted for use with probesenabled by the present invention. These procedures include restrictionfragment length polymorphism (RFLP), ligase chain reaction (LCR) orpolymerase chain reaction (PCR).

We began our investigations by considering whether there weredifferences between the chondrogenic/osteogenic differentiation factorsthat characterized calcifying (epiphyseal, scapular cartilage) andnon-calcifying (articular, nasal septum) cartilage tissues. It had beenpreviously established that tail tendon, achilles tendon, cartilage andskin matrices were devoid of chondrogenic/osteogenic activity(originally described as “transforming potency”) as measured in an invivo subcutaneous implantation model in rats (Reddi A. H., 1976,“Collagen and Cell differentiation” in Biochemistry of Collagen, eds.Ramachandran G. N. and Reddi, A. H., pp449-478, Plenum Press, New Yorkand London).

We confirmed the absence of chondrogenic or osteogenic activity in crude4 M guanidine HCl (GdnHCl) extracts of cartilage matrices, butunexpectedly discovered in vivo chondrogenic activity in the 0.15 M NaCleluate of the cartilage extracts after ion exchange chromatography. Thedevelopment of a unique extraction procedure (1.2 M GdnHCl and 0.5%CHAPs) followed by a heparin Sepharose affinity chromatography stepconfirmed the presence of in vivo chondrogenic activity in cartilaginoustissues. This was especially true in bovine articular and epiphysealcartilage. When the bioactive heparin Sepharose eluates (1M NaCl eluate)were further purified using previously established procedures, molecularsieve chromatography and Con A affinity chromatography steps followed bySDS polyacrylamide gel electrophoresis and gel elution, chondrogenicactivity was established. Implantation of 0.5 to 1 μg gel elutedmaterial resulted in in vivo chondrogenesis. Surprisingly, and incontrast to the bone matrix purified activity, none of the peptidesequences that were found in tryptic digests of the highly purifiedcartilage extracts corresponded to any of the known BMPs. However, thebiological activity present in the extracts was reminiscent of BMP-likeactivity by virtue of its loss of activity upon reduction andalkylation, its affinity for heparin Sepharose and Con A.

Although other materials and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.General references for methods that can be used to perform the variousnucleic acid manipulations and procedures described herein can be foundin Molecular Cloning: A Laboratory Manual (Sambrook et al. eds. ColdSpring Harbor Lab Publ. 1989) and Current Protocols in Molecular Biology(Ausubel et al. eds., Greene Publishing Associates andWiley-lnterscience 1987). The disclosures contained in these referencesare hereby incorporated by reference. A description of the experimentsand results that led to the creation of the present invention follows.

We initially discovered that an extract of cartilage possessed a uniquechondrogenic activity. In particular, we discovered that newbornarticular cartilage contained chondrogenic activities when assayed inthe in vivo subcutaneous implantation model. Using a procedure adaptedfrom that used for the isolation of BMPs from demineralized bone matrix,we partially purified this activity and thereby provided evidence forthe presence of BMP-like molecules in cartilage.

Example 1 describes the biochemical methods used to characterize achondrogenic activity present in bovine cartilage.

EXAMPLE 1 Characterization of Cartilage Derived Chondrogenic Activity

Articular (metatarsophalangeal joints), scapular and nasal cartilage(300 grams wet weight per tissue) were prepared from newborn calves.Epiphyseal cartilage was dissected from fetal bovine femurs (7-8months). The tissues were finely minced and homogenized with a Polytron(top speed, 2×30 seconds) in 20 volumes of 1.2 M GdnHCl, 0.5% CHAPS, 50mM Tris-HCl pH 7.2, containing protease inhibitors and extractedovernight at 4° C. as described by Luyten et al., in J. Biol. Chem.264:13377 (1989), which is hereby incorporated by reference. Thedisclosure of this article is hereby incorporated by reference. Sorgenteet al., (Biochem Biophys. Acta. 1972 282:441) disclosed these proceduresextract >90% of the lower molecular weight matrix while leaving most ofthe high molecular weight proteoglycans behind. The extracts wereconcentrated and exchanged with 6 M urea by diafiltration using anUltrasette™ (Filtron Technology Inc., MA) and loaded on a 0.5 L heparinSepharose (Pharmacia/LKB, NJ) column. Thereafter, the column was washedwith 5 bed volumes of 6 M urea, Tris HCl pH 7.4 with 0.15 M NaCl, andthen eluted with 2 vol 1 M NaCl in the same buffer. Chondrogenicactivity was assayed by reconstituting a portion of the eluate with 25mg of guanidine-insoluble collagenous residue of demineralized rat bonematrix according to procedures described by Luyten et al., in J. Biol.Chem. 264:13377 (1989). Implants were recovered after 10 days andalkaline phosphatase activity was measured as a biochemical indicator ofcartilage and/or bone formation. The specific activity was expressed asunits of alkaline phosphatase/mg of protein used for reconstitution inthe bioassay. Implants were also examined histologically for evidence ofcartilage formation using standard procedures known to those of ordinaryskill in the art.

Additional purification steps were also performed. The 1 M NaCl eluateof articular cartilage, which contained biological activity, wasconcentrated by diafiltration and loaded onto a Sephacryl S-200 HR gelfiltration column (XK 50/100, Pharmacia/LKB, NJ). After molecular sievechromatography, the bioactive fractions were pooled and exchanged with50 mM Hepes, pH 7.4, containing 0.15 M NaCl, 10 mM MgSO₄, 1 mM CaCl₂ and0.1% (w/v) CHAPS using Macrosep™ concentrators (Filtron Technology Inc.,Northborough, Mass.). The equilibrated sample was mixed with 1 ml Con ASepharose (Pharmacia-LKB) previously washed with 20 volumes of the samebuffer according to the procedure described by Paralkar et al., inBiochem. Biophys. Res. Comm. 131:37 (1989). After overnight incubationon an orbital shaker at 40° C., the slurry was packed into a disposable0.7 cm ID Bio-Rad column and washed with 20 volumes of the Hepes bufferto remove unbound proteins. Bound proteins were eluted with 20 volumesof the same buffer containing 500 mM methyl-D-mannopyronaside. Theeluate was concentrated to 200 μl using Macrosep™ concentrators.Macromolecules were then precipitated overnight with 9 volumes ofabsolute ethanol at 4° C. The precipitate was redissolved in 1 ml 6 Murea, Tris HCl pH 7.4. The bioactive bound protein was then mixed with2× Laemmli sample buffer (without reducing agents) and electrophoresedon a 12% preparative SDS/polyacrylamide gel. Gel elution of theseparated protein fractions and testing for biological activity wasperformed as described by Luyten et al., in J. Biol. Chem. 264:13377(1989). We also observed that, after reduction with dithiothreitol andalkylation with iodoacetamide, substantially all of thecartilage-forming activity contained in the protein sample was lost.

Results indicated that each of the crude extracts of the differentcartilaginous tissues (articular, nasal, scapular or epiphyseal) wereinactive when tested directly in the in vivo cartilage and bone inducingassay. This finding confirmed previously described results published byReddi in “Collagen and Cell differentiation” in Biochemistry of Collagen(eds. Ramachandran G. N. and Reddi, A. H., pp449-478, Plenum Press, NewYork and London (1976). However, after heparin affinity chromatography(Sampath et al., 1987 PNAS USA 84:7109), chondrogenic activity wasrecovered in the 1 M NaCl eluate from articular cartilage extracts. Anadditional molecular sieve chromatography step (S200) was required torecover chondrogenic activity from epiphyseal cartilage extracts.Similar results were obtained upon ion exchange chromatography usingDEAE Sephadex (0.15 M NaCl eluate). Significantly, no activity wasdetected in the extracts of the other cartilaginous tissues.

The highest specific activity was obtained for material derived fromarticular cartilage (1 U alkaline phosphatase/mg protein). This materialwas used for characterization of the bioactivity. Further purificationof the active fraction by molecular sieve chromatography on SephacrylS-200HR (specific activity 112 U/mg), and affinity chromatography onConcanavalin A (specific activity 480 U/mg), established the presence ofcartilage and bone inducing activity characteristic of the members ofthe BMP family. Gel elution experiments with the Con A bound bioactivefraction demonstrated that the activity resided between roughly 34 and38 kDa (specific activity of the gel eluted fractions was 2143 U/mg). Wehave also demonstrated that size separation by molecular sievechromatography can be used to purify biological activity in the 30-60kDa size range. In addition, loss of activity that was observedfollowing reduction and alkylation suggested that the bioactivity wasinduced either by a known or a new member(s) of the BMP family.

Given the demonstration that cartilage contained a BMP-like activity, weproceeded to isolate polynucleotides encoding the responsible proteins.Specifically, degenerate primers corresponding to conserved regions ofknown BMPs were designed. These primers were then employed to amplifypolynucleotides using reverse transcribed mRNA from articular cartilageas a template. These procedures ultimately led to the identification oftwo novel cDNAs, which we called CDMP-1 and CDMP-2.

Example 2 describes the methods used to amplify polynucleotidescorresponding to mRNAs that were expressed in cartilage tissue and thatexhibited at least weak sequence similarity to conserved regions of theBMP mRNAs.

EXAMPLE 2 PCR Amplification of cDNAs Encoding Cartilage-DerivedMorphogenetic Proteins

Total RNA from bovine articular chondrocytes (metatarsophalangealjoints) was extracted using a modified acid guanidine-phenol-chloroformmethod described by Chomczynski et al., in Anal. Biochem. 162:156 (1987)and by Luyten et al., in Exp. Cell. Res. 210:224 (1994). Poly(A)⁺ RNAwas isolated using magnetic beads (PolyATract™, Promega, Madison, Wis.).Four degenerate oligonucleotide primers corresponding to highlyconversed motifs in the C-terminal region of the BMPs were used; S1:5′-GGITGG(C/A)AIGA(C/T)TGGAT(A/C/T)(A/G)TIGC(A/C/G/T)CC-3′ (SEQ IDNO: 1) corresponding to amino acids [GW(Q/N)DWI(I/V)AP] (SEQ ID NO: 2);S2: 5′-GGITGG(A/T)(G/C)(I)GA(G/A)TGGAT(T/C/A)ATI(A/T)G(A/C/G/T)CC-3′(SEQ ID NO: 3) corresponding to amino acids [GWSEWIISP] (SEQ ID NO: 4);AS1: 5′-A(A/G)A/G)GT(C/T)TG(A/C/G/T)AC(A/G)AT(A/G)GC(A/G)TG(A/G)TT-3′(SEQ ID NO: 5) corresponding to amino acids [NHAIVQTL] (SEQ ID NO: 6);AS2: 5′-CAI(C/G)C(A/G)CAI(G/C)(A/C/T)I(C/T)(C/G/T)IACIA(C/T)CAT-3′ (SEQID NO: 7) corresponding to amino acids [M(V/I)V(E/R)(G/S/A)C(G/A)C] (SEQID NO: 8). Nucleotides in parenthesis denote sites of degeneracy and Idenotes inosine. First strand cDNA synthesis was performed using 1 μgPoly(A)⁺ or 5 μg total RNA with oligo dT, random hexanucleotide primers,or the antisense degenerate primers, AS1 and AS2. Successful PCRamplifications were performed with the degenerate sense primers, S1 orS2 in combination with the AS 1 antisense primer were performed usingconditions described by Wharton et al., in PNAS USA 88:9214 (1991). Thereaction products were electrophoresed on 1.2% agarose gels, and DNAfragments of appropriate sizes were excised and purified using the MagicPCR Prep DNA purification system (Promega, Madison, Wis.).Reamplification was performed with the same primers and each PCR productwas subcloned into the PCR II vector using the TA Cloning™ System (InVitrogen Corporation, San Diego, Calif.). Results of RT-PCR usingpoly(A)⁺ RNA isolated from newborn bovine articular cartilage astemplate and sets of degenerate oligonucleotide primers (S1/AS1 andS1/AS2) yielded amplification products of 120 bp and 280 bp.

Subcloned inserts were sequenced according to the dideoxy DNA sequencingmethod of Sanger et al., (PNAS USA 1977 74:5463). Both DNA strands weresequenced using Sequenase Version 2.0 DNA polymerase according tomanufacturer's instructions (USB, Cleveland, Ohio) with at leasttwo-fold redundancy. Confirmatory data in ambiguous regions wereobtained by automated thermal cycle sequencing with an AppliedBiosystems Model 370A sequencer and by using 7-deaza-GTP (USB,Cleveland, Ohio). The sequencing data were obtained from restrictionfragments subcloned into pBluescript (Stratagene, La Jolla, Calif.)using either M13 forward and reverse primers or syntheticoligonucleotide primers.

The results from a computer-assisted search of the nucleic acid sequencedatabases indicated the cloned inserts encoded BMP-2, -6, BMP-7 (OP-1),and several other BMP-like sequences. Identification of these lattergene fragments led us to isolate larger cDNAs that included the entireprotein coding region of the transcript. The availability of such clonesfacilitated both a more precise analysis of the encoded BMP-like proteinand permitted studies aimed at localizing the expression of these genes.Thus, cloned inserts having novel BMP-like sequences were isolated,radiolabeled and used to screen both human and bovine articularcartilage cDNA libraries.

Example 3 describes the methods used to isolate human and bovine cDNAsthat corresponded to a segment of one of the BMP-like gene segments thatwere amplified from cartilage mRNA templates.

EXAMPLE 3 Library Screening

A 120 bp PCR fragment encoding part of the C-terminal domain of novel Blike genes (dashed line, FIG. 1) was used to screen two cDNA libraries.One library, from adolescent human articular cartilage poly(A)⁺ RNA(kindly provided by Dr. Bjorn Olsen, Harvard, Boston, Mass.), was primedwith oligo dT and constructed in the λgtl 1 vector. The other was abovine oligo dT and random primed articular cartilage cDNA libraryconstructed in the UNIZAP®XR vector (Stratagene, La Jolla, Calif.).Approximately 1×10⁶ plaques from each library were screened by standardprocedures. Hybridizations were performed for 20 hours at 42° C. in6×SSC, 1× Denhardt's solution, 0.01% tRNA, 0.05% sodium pyrophosphateand the membranes (DuPont 137 mm nylon membranes, New England Nuclear,MA) were washed to final stringency of 6×.SSC, 0.1% SDS at 55° C. for 20minutes.

Thus, cloned inserts having novel BMP-like sequences were isolated,radiolabeled and used to screen both human and bovine articularcartilage cDNA libraries. Six clones were isolated from the human cDNAlibrary. The sizes of the EcoRI inserts (2.1 kb) and their restrictionmaps were found to be identical for all six clones. One clone was usedfor nucleotide sequencing. An open reading frame encoding a BMP relatedprotein, designated CDMP-1, was identified. It appeared that the humancDNA clone lacked the coding region for the first methionine and signalpeptide. The 5′ end of the human CDMP-1 was subsequently obtained from ahuman genomic clone isolated from a library constructed in the EMBL-3vector (Clontech, Palo Alto, Calif.). The 5′ end of human CDMP-1contained a consensus translation initiation sequence disclosed by Kozak(J. Biol. Chem. 1991 266:19867) immediately followed by a putativetransmembrane signal sequence described by Von Heijne (Nucl. Acids Res.1986 14:4683). The nucleotide sequence and the translation of the openreading frame of CDMP-1 are presented in FIG. 1 and deposited atAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110, USA, as PTA-2595, on Oct. 16, 2000. This depositwas made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Applicant and ATCC which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC § 122 and the Commissioner's rules pursuant thereto (including37 CFR § 1.14). Availability of the deposited strain is not to beconstrued as a license to practice the invention in contravention of therights granted under the authority of any government in accordance withits patent laws.

As shown in the figure, the CDMP-1 protein was predicted to have 500amino acids, to consist of a pro-region of 376 amino acids, a typicalcleavage site (Arg-Xaa-Xaa-Arg/Ala) (SEQ ID NO: 9), and a C-terminaldomain of 120 amino acids containing the seven highly conservedcysteines characteristic of the TGF-β gene family. A single N-linkedglycosylation site is located in the pro-region (marked. by an asteriskin the figure). A putative signal peptide is underlined in bold. Atermination codon (TGA) is shown in the 5′ untranslated region. The bolddashed underline indicates the fragment obtained by RT-PCR that wassubsequently used to screen cDNA libraries. The 13 amino acid peptideused to raise polyclonal antibodies in rabbits is underlined. A verticalarrowhead marks the boundary between the sequence obtained from genomicDNA and cDNA.

Two clones with inserts of 2.8 kb were isolated from a bovine articularcartilage cDNA library. Both clones were sequenced and the open readingframe was found to encode another novel TGF-β related protein,designated CDMP-2. The CDMP-2 cDNA and predicted protein sequences arepresented in FIG. 2. As shown in the figure, the open reading framecontained a putative proteolytic processing site (boxed), preceding a120 amino acid mature C-terminal region containing seven highlyconserved cysteines. The 5′ end with the first methionine and signalpeptide were missing. The product obtained by RT-PCR (bold dashedunderline) was used to screen a bovine cDNA articular cartilage library.The ApaI sites used to release a cDNA fragment for hybridizationexperiments are underlined. At the 5′ end, the pro-region lacked thefirst methionine and signal peptide. The mature C-terminal domain of 120amino acids showed 82% identity with CDMP-1.

Alignment of the carboxy terminal domains of CDMP-1 and -2 with othermembers of the BMP family revealed an amino acid identity of about 50%with BMP-5, BMP-6 and OP-1 (BMP-7). These results suggested that CDMP-1and CDMP-2 are members of a new subfamily.

The amino acid sequence similarity between the human CDMP-1 and bovineCDMP-2 proteins prompted us to further investigate conservation of theCDMPs across different species. In particular, we employed a PCRamplification protocol to isolate CDMP cDNA sequences from a variety ofspecies. Based on alignments of the predicted proteins encoded by thesecDNAs, we identified a highly conserved amino acid sequence spanning 31residues. Only 5 amino acid positions within this sequence showedvariability. All remaining positions were identical for all isolates. Asdisclosed in the following Example, even the 5 variable positions showeda high degree of conservation. This structural conservation likelyrepresents a functional domain that is characteristic of the CDMP familyof proteins. Those of ordinary skill in the art will appreciate thatsuch extraordinary amino acid sequence conservation is indicative of afunctional domain. We therefore believe the consensus amino acidsequence presented in the following Example is critical to thebiological activity of the CDMPs.

Example 4 describes the procedures used to identify an amino acidconsensus sequence that characterizes the CDMPs from several differentspecies.

EXAMPLE 4 Identification of a Highly Conserved Consensus Sequence inCDMP Proteins

RNA isolated from chicken sternal cartilage, bovine articular cartilageand human articular cartilage was employed as the template in RT-PCRprotocols using the primers S1 and AS1 and procedures described underExample 2. Genomic DNA isolated from Xenopus and zebrafish was also usedas the template for amplification of related gene sequences in a PCRprotocol that employed the same primer sets. Amplified DNA fragmentswere subcloned according to standard procedures. The inserts fromvarious isolates were sequenced by standard dideoxy chain terminationprotocols. Aligned segments of the predicted proteins encoded by thecloned cDNAs are presented in FIG. 4.

Results of the protein alignments clearly indicated that CDMP familymembers from several species shared a common amino acid sequence motifin the region of the proteins encoded by the amplified cDNA segments. Ofthe 31 amino acid positions presented in FIG. 4, all but 5 were occupiedby identical amino acid residues for all of the isolates. The variableamino acids were located at positions 3, 7, 11, 16 and 18. Position 3was occupied either by I, M or V. Position 7 was occupied by either D orE, both of which have acidic side groups. Position 11 was occupied byeither Y, F or H. Position 16 was occupied by L or V, and position 18was occupied by D or E. The consensus deduced from this alignment was:W-I-(I/MJV)-A-P-L-(D/E)-Y-E-A-(Y/F/H)-H-C-E-G-(LNV)-C-(D/E-)-F-P-L-R-S-H-L-E-P-T-N-H-A(SEQ ID NO: 15). This consensus sequence is slightly broader than theone shown in FIG. 4, as it encompasses all the variations observed inthe sequenced polynucleotides. The consensus sequence in the figureindicates predominating amino acids.

We believe that biologically active CDMPs will possess this highlyconserved amino acid sequence motif. Proteins having different aminoacids in the variable positions in the consensus will likely representnovel family members having distinct functions. We also believe thatpolynucleotide hybridization probes or PCR primers designed based onthis conserved protein motif can be used to isolate cDNAs encoding CDMPfamily members or related proteins.

Southern analyses were also carried out to investigate possible sequenceconservation across species and to localize the CDMP-1 gene to aparticular chromosome.

Example 5 describes the Southern blotting protocols used to detect DNAsequences corresponding to the CDMP-1 cDNA.

EXAMPLE 5 Genetic Mapping of CDMP-1

Southern hybridization was performed using the evolutionary relatednessblot (Bios Laboratories, New Haven, Conn.) under conditions recommendedby the manufacturer. The panel of EcoRI-digested genomic DNAs includedhuman (homo sapiens), mouse (Mus musculus), chicken (Gallus domesticus),frog (Xenopus laevis), lobster (Homarus americanus), mussel (Mytilusedulis), fish (Tautoga onitis), fruit fly (Drosophila melanogaster),nematode (Caenorhabditis elegans), yeast (Saccharomyces cerevisiae) andbacteria (E. coli). The 2.1 kb CDMP-1 EcoRI fragment originally obtainedfrom the cDNA library was used as a probe, and the blot was washed to afinal stringency of 0.4×SSC, 0.1% SDS, at 55° C.

Results from these Southern analyses using the original 2.1 kb humanCDMP-1 cDNA probe (starting from amino acid position 40), showed 5.9 and2.6 kb bands in humans and strong hybridization in both mouse andchicken. Fainter bands were seen in fish, frog and lobster after 5 daysautoradiographic exposure. No hybridization was detected to DrosophilaDNA.

The 2.1 kb ApaI fragment of CDMP-1 was used as a hybridization probe onSouthern blots to type mouse genomic DNAs from two genetic crosses:(NFS/N or C58/J×M. m. musculus)×M. m. musculus (see Joseph et al., 1990Mol. Immunol. 30:733) and (NFS/N×M. spretus)×M. spretus or C58/J (seeAdamson et al., 1990 Virology 183:778). DNAs from these crosses havebeen typed for over 650 markers including the Chr 2 markers Snap(synaptosomal associated protein 25), Psp (parotid secretory protein),Emv15 (ecotropic murine leukemia virus 15), Src (src oncogene), and Cd40(cluster designation 40). Probes for these markers and restrictionfragment length polymorphisms used to type these crosses have beendescribed by Joseph et al., in Mol. Immunol. 30:733 (1990) and byGrimaldi et al., in J. Immunol. 149:3921 (1992). Src was typed using amouse Src probe obtained from E. Rassart (U. Quebec, Montreal) followingXbaI digestion in the musculus cross and BamHI digestion in the spretuscross.

Results from Southern blotting with the 2.1 kb cDNA described aboveidentified EcoRI fragments of 7.1 and 2.0 kb in M. m. spretus and M. m.musculus and 6.8 and 3.2 kb in NFS/N and C58/J.

Inheritance of the polymorphic fragments in the progeny of the twocrosses used for mapping was compared with inheritance of over 650markers previously mapped to all 19 autosomes and the X chromosome. Thegene encoding CDMP-1 was found to be linked to markers on Chr 2 justproximal to Src. The closest linkage was observed with Psp and Emv15. Norecombination was observed between Cdmpl and Psp in the 100 mice typedfor both markers indicating that these genes are within 3.0 cM at the95% confidence level. Similarly, the absence of recombination betweenGdf5 (Storm et al., 1994 Nature 368:639) and Cdmp1 in 125 mice suggestedthese genes colocalized within 2.4 cM. This map location suggested closeproximity to the brachypodism locus (bp). A genetic map that presentsthe localization of CDMP-1 on chromosome 2 is shown in FIG. 3.Recombination fractions are given to the right of each map of thediagram for each adjacent locus pair or cluster. Numbers in parenthesisrepresent the percent recombination and standard error calculated asdescribed by Green in Genetics and Probability in Animal BreedingExperiments, Oxford University Press, New York (1981). The map on theleft is an abbreviated version of the Chr 2 Committee Map disclosed bySiracusa et al., in Mammal Genome 4:S31 (1993), and shows the maplocation of bp relative to the other markers typed in the crosses usedhere.

The brachypodism (bp mice) disorder is characterized by a distinctshortening of the limbs without other tissue abnormalities. The defecthas previously been attributed to lack of production of a chondrogenicsignal by mesenchymal cells at the time of chondrogenesis (Owens et al.,1982 Dev. Biol. 91:376). During the course of our investigation, anindependent study by Storm et al. (Nature 368:639, 1994) described theisolation of the mouse CDMP-1 homolog, called Gdf-5, and established itslinkage to the brachypodism (bp) mutation. The types of mutationsobserved in bp mice were found to be effective null-mutations for thegene encoding Gdf-5/CDMP-1. The pattern of expression of CDMP-1throughout the cartilaginous core observed during human embryonic longbone development, coupled with the bp mutation in mice, indicated thatits primary physiological role was most likely at the stage of earlychondrogenesis and chondrocyte differentiation in the developing limb.

The foregoing results indicated the CDMP-1 and CDMP-2 cDNAs were novel,exhibited moderate sequence conservation across species as judged byevolutionary hybridization studies and that the CDMP-1 gene localized tomouse chromosome 2. We proceeded to examine the pattern of CDMPexpression at the mRNA level.

Example 6 demonstrates the methods used to determine the pattern of CDMPmRNA expression.

EXAMPLE 6 CDMPs are Predominantly Expressed in Cartilage DuringPostnatal Life

Equal amounts of poly(A)⁺ RNA (2 μg) from bovine criocoid and articularcartilage were electrophoresed on 1.2% agarose-formaldehyde gels andthen transferred to Nytran membranes (Schleicher and Schuell, Kenne,N.H.) according to standard laboratory procedures. Multiple TissueNorthern blots were obtained from Clontech (Palo Alto, Calif.). Themembranes were prehybridized for 3 hours at 42° C. in hybridizationbuffer (5×SSPE, 5× Denhardt's solution, 50% formamide, 1% SDS and 100μg/ml freshly denatured salmon sperm DNA). Hybridizations with [³²P]dCTPlabeled probes, having specific activities of at least 1×10⁹ CPM/μg,were performed overnight under the same conditions as theprehybridization. Probes included the cDNA probe for humanglyceraldehyde-3-phosphate dehydrogenase (1.1 kb, G3PDH (Clontech, PaloAlto, Calif.), an ApaI fragment (bp 470-1155) of CDMP-1, and an Apalfragment (bp 194-677) of CDMP-2. The CDMP-1 and CDMP-2 probes werechosen to avoid the highly conserved carboxy-terminal domain, therebyminimizing the potential for cross hybridization with other members ofthe gene family. Following hybridization, the filters were washed to afinal stringency of 55° C., 0.4×SSC, 0.1% SDS. The mRNA expressionlevels were quantified using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

Results from Northern analyses of a number of postnatal tissuesindicated that CDMP-1 could predominantly be detected in newbornarticular and cricoid cartilage. In both cases a single mRNA transcriptof approximately 3 kb was observed. The CDMP-1 mRNA was not detected inpancreas, kidney, skeletal muscle, liver, lung, placenta, brain orheart. In contrast, BMP-3 and BMP-7 transcripts were detected insubsequent hybridizations of the same blots in mRNA samples from lung,kidney, brain and small intestine. This finding was consistent withprevious results disclosed by Vukicevic et al., (J. Histochem. Cytochem.42:869, 1994). CDMP-2 mRNA was detected in postnatal bovine articularand cricoid cartilage as a 4.6 kb mRNA band. After prolonged exposure,weak hybridization signals were detected at 4.6 kb and 4.0 kb in mRNAfrom colon and small intestine, skeletal muscle and placenta.

Two other procedures were used to localize and visualize expression ofthe CDMP-1 and CDMP-2 gene products. These approaches relied ondetection of mRNA and protein in tissue sections prepared for analysisby microscopy.

Example 7 describes the methods used to demonstrate the preferentialexpression of CDMPs during human embryogenesis.

EXAMPLE 7 CDMPs are Preferentially Expressed in the Cartilaginous Coresof Long Bone During Human Embryogenesis

In Situ Hybuidization

Tissues from human embryos were obtained after pregnancy termination atfrom 5 to 14 weeks of gestation. Embryo age was estimated in weeks (W)on the basis of crown-rump length (CRL) and pregnancy records of theconceptual age. They were fixed in 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.2), embedded in paraffin, sectioned serially at5-7 μm, and mounted on silanated slides. The tissues used in the presentstudy were obtained from legally sanctioned procedures performed at theUniversity of Zagreb Medical School. The procedure for obtaining thehuman autopsy material was approved and controlled by the InternalReview Board of the Ethical Committee at the School of Medicine,University of Zagreb and Office of Human Subjects Research (OHSR) at theNational Institutes of Health, Bethesda, Md. In situ hybridization wasdone as described by Vukicevic et al., (J. Histochem. Cytochem. 42:869,1994) and by Pelton et al. (Development 106:759, 1989). Briefly,sections were incubated overnight at 50° C. in a humidified chamber in50% formamide, 10% dextran sulfate, 4×SSC, 10 mM dithiothreitol, 1×Denhardt's solution, 500 μg/ml of freshly denatured salmon sperm DNA andyeast tRNA with 0.2-0.4 ng/ml ³⁵S labeled riboprobe (1×10⁹ CPM/μg). ApaIfragments of CDMP-1 and of CDMP-2 (described above) from the pro region,subcloned in both sense and anti-sense direction into pBluescript II(SK)⁺ vector (Strategene, Calif.), were used as transcription templates.Riboprobes were then prepared using T7 RNA polymerase (Sure Site Kit,Novagen, Madison, Wis.) according to the manufacturer's instructions andused with and without prior alkaline hydrolysis. After hybridization,the sections were washed as described by Lyons et al., in Development109:833 (1990), to a final stringency of 0.1×SSC, 65° C. for 2×15minutes. After dehydration through a graded ethanol series containing0.3 M ammonium acetate, slides were covered with NTB-2 emulsion (Kodak)and exposed between 1 and 3 weeks. After development, the slides werestained with 0.1% toluidine blue, dehydrated, cleared with xylene andmounted with Permount.

Immunostaining

A polyclonal antibody to the peptide QGKRPSKNLKARC (SEQ ID NO: 10)(amino acids 388-400; prepared by Peptide Technologies, Gaithersburg,Md.), which belongs to the mature secreted protein of CDMP-1, was raisedin rabbits. Before immunization, the peptide was conjugated to Imject™Malemide Activated Keyhole Limpet Hemocyanin (Pierce, Rockfor, Ill.).Searches performed using the BLAST (Altschul et al., 1990 J. Mol. Biol.215:403) network service available through the National Center forBiotechnology Information indicated that the peptide does not showsequence identity with any known protein or BMP. The embryonic tissuesections were stained as recommended by the manufacturer usingimmunogold as a detection system (Auroprobe L M; Janssen, Belgium) andcounterstained with 0.1% toluidine blue. The primary antibody (crudeantiserum) was used at a concentration of 15 μg/ml in PBS with 0.5%bovine serum albumin (BSA) for 1 hour. In the controls, the primaryantibody was replaced with BSA, normal rabbit serum, or secondaryantibody alone.

Results indicated that, at 6 weeks of gestation, CDMP-1 transcripts weredetected in precartilage condensations within the developing limbs. At7.5-8.5 weeks of gestation, CDMP-1 mRNA expression was found in thecartilaginous cores of long bones, including the articular surfaces. Inareas of active cartilage degradation and bone matrix formation, CDMP-1expression was also detected in hypertrophic chondrocytes. Remarkably,no expression was detected in the axial skeleton and only low mRNAlevels were observed in other tissues, such as distal convoluted tubulesof the developing kidney, brain and placenta. Immunohistochemicalstaining indicated that CDMP-1 protein colocalized with the mRNA.However, in addition to the sites of transcription, the protein was alsofound in the surrounding cartilaginous matrix and in osteoblast-likecells from the primary ossification centers of long bones.

Between 9 and 10 weeks of gestation, CDMP-2 expression was predominantlylocalized in the more mature and hypertrophic chondrocytes in regions ofinvasion by blood vessels through the periosteal bony collar of thedeveloping long bone. Again, as for CDMP-1, no hybridization wasdetected in the vertebral bodies in the corresponding sections andstages of human embryonic development. Low expression levels weredetected in the periosteum.

The expression pattern of CDMP-2 suggests it is involved in the terminaldifferentiation of chondrocytes (hypertrophic and mineralizing) and atthe earliest stages of endochondral bone formation, includingangiogenesis and osteoblast differentiation. In addition, the relativelyhigh levels of expression (detectable in total RNA blots) in postnatalcartilage suggest possible roles in the maintenance and stabilization ofthe cartilage phenotype after birth.

We have also designed experiments aimed at determining whether all ofthe chondrogenic activity contained in cartilage extracts can beattributed to the proteins encoded by the CDMP-1 and CDMP-2 cDNAs. Ourapproach involves the production and use of neutralizing antibodiesusing synthetic peptides or recombinant CDMP-1 and CDMP-2 proteins asimmunogens. Antibodies raised against these peptides or proteins will betested for their ability to deplete cartilage extracts of chondrogenicactivity. If antibodies specific for the recombinant proteins fail todeplete the extracts of cartilage-forming activity, then residualactivity will be due to factors within the extract that are separatefrom proteins encoded by the CDMP-1 and CDMP-2 proteins. Alternatively,if antibodies raised against the peptides or recombinant proteins canremove cartilage-inducing activity from the extracts, this will confirmthat CDMP-1 and/or CDMP-2 must be responsible for the active agentscontained in the extracts.

Example 8 describes the methods that will be used to raise antibodiesagainst synthetic peptides and recombinant CDMP-1 and CDMP-2 proteins.Antibodies produced in this fashion will be tested for their ability todeplete extracts containing CDMP activity.

EXAMPLE 8 Production and Use of Anti-CDMP Antibodies

Specific monoclonal and polyclonal antibodies will be raised againstpeptides designed from the mature protein of the CDMPs. Preferentially,the region between the protein cleavage site and the first cysteine ofthe CDMP-1 and CDMP-2 proteins will be used to design the peptides. Inaddition, the cDNAs encoding the mature region of the CDMPs will besubcloned in the bacterial pET expression vector, and expressed asmonomers in the bacterial expression system. The protein expressed inthis system will be used to raise additional antibodies, and todetermine the immunoreactivity of the various antisera in Western blots.The bacterially expressed monomers will be refolded into biologicallyactive dimers using standard protocols. This approach may afford anothersource of recombinant protein.

The antisera obtained in this fashion will be used to further establishthe synthesis of the CDMPs by chondrocytes in vivo and in vitro, and tolink the cloned CDMPs to the chondrogenic activity found in cartilageextracts. Conditioned media obtained from chondrocyte cultures andpartially purified chondrogenic cartilage extracts after heparinsepharose affinity chromatography, molecular sieve chromatography andCon A chromatography, will be analyzed for the presence of CDMPs byWestern blot analysis. Due to the possible heterogeneity of the highlypurified chondrogenic cartilage extracts, the antibodies will be used toreduce or deplete the chondrogenic/osteogenic activity in purifiedfractions in a standard immunoprecipitation experiment.

An important aspect of our invention regards the production and use ofrecombinant proteins that possess the biological activities of theCDMPs. The following Example describes methods and results thatillustrate the production of recombinant CDMP-1 and CDMP-2 intransfected 293 cells, COS-1 cells, and CHO-1 cells. We discovered that293 cells express BMP-7 that could conceivably contaminate preparationsof recombinant CDMPs. To avoid possible ambiguities in theinterpretation of our results, recombinant CDMP-1 produced in COS-1cells was used to demonstrate cartilage forming activity. Although theproduction of recombinant CDMPs in this fashion was rather inefficient,the key finding illustrated by our results was that recombinant proteinhad the desired cartilage-forming activity. Unexpectedly, and incontrast to the related BMPs, recombinant CDMP-1 induced cartilageformation without noticeable bone formation.

Example 9 describes the procedures used to produce recombinant CDMPproteins. The results presented in the Example confirm that therecombinant cartilage-derived proteins stimulated cartilage formation.

EXAMPLE 9 Production of Recombinant CDMPs and Assessment of Bioactivity

Full length CDMP-1 was subcloned into the mammalian expression vectorpcDNA3 (Invitrogen Corporation, San Diego, Calif.) containing thecytomegalovirus early gene promotor and other elements required forexpression in mammalian cells. COS 1 cells were cultured in Opti-MEM I(Gibco/BRL, Gaithersburg, Md.) in the presence of 5% fetal bovine serumand antibiotics. The cells were grown to approximately 70-80% confluencyin 150 mm dishes and transfections of the respective plasmids (20 μgplasmid) were carried out by the calcium phosphate method using thetransfection MBS mammalian transfection kit (Stratagene, La Jolla,Calif.). The cells were incubated with the calcium phosphate-DNA mixturefor 3 hours at 35° C. Supernatants were removed and the plates werewashed 3 times with PBS. 15 ml of Opti-MEM I (serum reduced medium) wasadded in the absence of serum, and the dishes were incubated overnight.Transfection efficiencies were tested by transfection of a controlplasmid, pCMVβ-gal and cell extracts were assayed for β-galactosidaseactivity. Conditioned media were collected at 24 hour intervals for aperiod of 96 hours. The pooled media was centrifuged to remove celldebris and then concentrated using Mascrosep 10 concentrators (FiltronTechnology Inc., Northborough, Mass.). Further purification ofrecombinantly expressed protein was performed as described in precedingExamples. In one exemplary procedure, the conditioned media was adjustedto 4 M urea, 25 mM Tris HCl (pH 7.0) and applied to a heparin Sepharosecolumn. The column was washed with the same buffer containing 0.1 MNaCl, and eluted with 1 M NaCl. The heparin Sepharose unbound and elutedfractions were assayed for biological activity as described by Luyten etal., in J. Biol. Chem. 264:13377, 1989.

Biological activity of the recombinantly expressed protein wasinvestigated using in vitro and in vivo chondrogenic/osteogenic assays.For the in-vivo assay, fractions containing the CDMPs were precipitatedwith ethanol, or dried onto a carrier such as bone matrix residue(mainly collagen type I particles) and cartilage matrix residue(cartilage tissue after extraction with chaotropic agents, andpowderized to particles with a size of 75-400 μm). The dried pellet(about 25 mg) was implanted subcutaneously in rats. Implants wereharvested after 11 and 21 days, and analyzed forchondrogenesis/osteogenesis using alkaline phosphatase determinations.Histological analysis of recovered samples was also performed usingtoluidine blue, alcian blue and safranin O staining. Results obtainedusing the recombinant CDMP-1 produced in COS-1 cells revealedchondrogenic activity in this in vivo assay. Significantly, noosteogenic activity was observed in any of the recovered samples.Osteogenic activity would ordinarily have been observed if the sameprocedures had been carried out using recombinant BMPs. This differencehighlighted the unique properties of recombinant CDMP-1.

Future in vitro chondrogenic experiments will be performed to determinethe precursor cells responsive to the CDMPs. Undifferentiated (10T1/2cells, bone marrow stromal cells, mesenchymal stem cells) and alreadycommitted skeletal cells (limb bud cells, perichondrial or periostealcells, fetal epiphyseal chondroblasts, and chondrocytes) will betransfected with the cDNAs or treated with recombinantly expressed CDMPsto evaluate the stage of differentiation associated with thechondrogenic activity of the CDMPs.

Future in vivo chondrogenic experiments will be directed to expressionof large quantities of CDMP-1 and CDMP-2 by stable transfectants. Wecontemplate the use of hybrid expression constructs in which thepro-region of one BMP family member (for example BMP-2) is operationallylinked to the regions encoding the mature CDMPs. We also anticipate invivo assays based on implantation in other sites, apart fromsubcutaneous implantation, which may reveal distinct or superiorbiological activities of the CDMPs. For example, we anticipateimplantation in the synovial cavity may have utility in such assays.

The CDMPs disclosed in the present invention have important applicationsin the repair of cartilage defects. We contemplate two generalapproaches for this type of therapy. In the first place, the CDMPs areused as lineage-specific growth factors for the ex vivo expansion ofchondrocytes isolated from a donor who requires therapeuticintervention. Following expansion, these cells can be reimplanted into acartilage lesion in the donor, whereafter repair of cartilage will takeplace. In a different scenario, CDMPs are introduced into a cartilagelesion. For example, a composition containing an appropriate CDMP ormixture of CDMPs can be implanted into a lesion for the purpose ofstimulating in vivo chondrogenesis and repair of cartilage. The CDMPscan be combined with any of a number of suitable carriers. Anappropriate carrier can be selected from the group comprising fibringlue, cartilage grafts, and collagens. An implantable mixture can beintroduced into the site of a lesion according to methods familiar tothose having ordinary skill in the art. In one application, wecontemplate that periosteal synovial membrane flap of tissue or inertmaterial can be impregnated with CDMPs and implanted for cartilagerepair.

Example 10 illustrates one application of the CDMP preparationsdescribed above. Specifically, the following Example describes the useof CDMPs to facilitate repair of cartilage in the knee joint.

EXAMPLE 10 Treatment of Deep Knee Defects with Cartilage-DerivedMorphogenetic Proteins

A young patient having a large defect in the articular surface of theknee joint is identified. According to standard surgical procedures, aperiosteal flap is obtained from the bone beneath the joint surface ofrib cartilage. The tissue flap is pre-soaked in an extract containingCDMPs or alternatively in a solution containing recombinant CDMPs. Theperiosteal flap treated in this way is then attached over the lesion inthe articular surface of the knee joint by a sewing procedure, forexample using resolvable material. The joint is then closed. The jointis injected with a solution containing CDMPs dissolved or suspended in apharmacologically acceptable carrier to maintain the chondrogenicprocess. Injection is continued until the monitoring physician indicatesrepair of the cartilage is complete. The patient notices markedly lessjoint pain as the cartilage repair process progresses. Exam byarthroscopy indicates repair of the lesion within several weeksfollowing the initial procedure.

We also contemplate gene therapy protocols based on expression of CDMPcDNAs or genomic constructs as a way of facilitating in vivo cartilagerepair. Diseases such as chondromalacia or osteoarthritis are examplesfor which such gene therapy protocols are contemplated. Therapy may beachieved by genetically altering synoviocytes, periosteal cells orchondrocytes by transfection or infection with recombinant constructsthat direct expression of the CDMPs. Such altered cells can then bereturned to the joint cavity. We contemplate that gene transfer can beaccomplished by retroviral, adenoviral, herpesvirus and adeno associatedvirus vectors.

Both in vivo and ex vivo approaches are anticipated for continuouslydelivering CDMPs for the purpose of retarding ongoing osteoarthriticprocesses and for promoting cartilage repair and regeneration. Inaddition, one might employ inducible promoter constructs (e.g. undertranscriptional control of a dexamethasone promoter) in gene therapyapplications of the present invention. A combined approach toosteoarthritis therapy may have particular advantages. For example,CDMP-2 could be continuously expressed to support the integrity of thearticular surface. An inducible construct could be employed to expressCDMP-1 so that chondrogenesis could be accelerated at the time of moreaggressive destruction.

The foregoing experimental results and characterization confirmed theCDMP-1 and CDMP-2 isolates belong to the TGF-β superfamily. Based on thehigh percentage identity of their C-terminal domains, CDMP-1 and CDMP-2can be classified as members of a novel subfamily. Although CDMP-1 andCDMP-2 were identified in two different species (human and bovine), theyrepresent distinct genes since the sequences of their pro-regions aresignificantly divergent.

Several BMPs have now been implicated in early skeletal development,including BMPs-2, -4, -5, -7 and CDMP-1 (GDF-5). Other members, such asBMPs-3, -6, -7 and CDMP-2, may be involved in later stages of skeletalformation (13, 15). The role of the BMPs in early development could bechemotactic, mitogenic or inductive. Their function in later stages ofskeletal development might be promotion of differentiation andmaintenance of the established phenotype. The availability of mousestrains with null mutations in specific BMP members, such as theshort-ear mice (Bmp5) and the bp mice (Cdmp1/Gdf5), allows analysis ofthe specific contributions of the respective members in each of thestages of skeletal development.

The absence of expression of both CDMP-1 and CDMP-2 in the axialskeleton has implications for models of skeletal development. Forexample, the bp mice have disturbed limb development but a normal axialskeleton. This is the first evidence that the developmental mechanismsand differentiation pathways of the vertebral bodies are distinct fromthose of the peripheral skeletal elements. Further, this indicates thebasic form and pattern of the skeleton are likely to be determined by anumber of BMP-like signaling molecules.

1. A purified cartilage extract that stimulates local cartilageformation when combined with a matrix and implanted into a mammal, saidextract being produced by a method comprising: (a) obtaining cartilagetissue; (b) homogenizing said cartilage tissue in the presence ofchaotropic agents under conditions that permit separation of proteinsfrom proteoglycans; (c) separating said proteins from saidproteoglycans; and (d) obtaining said proteins.
 2. The extract of claim1, wherein said extract is obtained by a method in which step (c)comprises use of a sepharose column.
 3. The extract of claim 1, whereinsaid extract is obtained by a method which additionally comprises thesteps of separating said proteins on a molecular sieve and obtainingthose proteins having a molecular weight between 30 kDa and 60 kDa. 4.The extract of claim 1, wherein said extract is an extract of articularcartilage.
 5. The extract of claim 1, wherein said extract is an extractof epiphyseal cartilage.
 6. A method of preparing a partially purifiedarticular cartilage extract having chondrogenic activity, comprising thesteps: (a) obtaining cartilage tissue; (b) homogenizing said cartilagetissue in the presence of chaotropic agents under conditions that permitseparation of proteins from proteoglycans; (c) separating said proteinsfrom said proteoglycans; and (d) obtaining said proteins.
 7. The methodof claim 6, wherein step (c) comprises use of a sepharose column.
 8. Themethod of claim 7, wherein step (c) comprises isolating the proteinsthat bind heparin Sepharose in the presence of 0.15 M NaCl but not inthe presence of 1 M NaCl.
 9. The method of claim 6, additionallycomprising the steps of separating said proteins on a molecular sieveand obtaining those proteins having a molecular weight between 30 kDaand 60 kDa.
 10. An isolated DNA molecule encoding a protein havingchondrogenic activity in vivo but substantially no osteogenic activityin vivo, said molecule having a nucleotide sequence that can hybridizeto a polynucleotide having a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11 and SEQ ID NO: 12 at 55° C. with 0.4×SSC and0.1% SDS.
 11. The isolated DNA molecule of claim 10, wherein saidmolecule has a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13 and SEQ ID NO:
 14. 12. A recombinant protein havingchondrogenic activity in vivo but substantially no osteogenic activityin vivo, wherein said protein has an amino acid sequence selected fromthe group consisting of SEQ ID NO: 13 and SEQ ID NO:
 14. 13. A method ofstimulating cartilage formation in a mammal, comprising the steps: (a)supplying cartilage-derived morphogenetic proteins having in vivochondrogenic activity; (b) mixing said partially purified proteins witha matrix to produce a product that facilitates administration of saidpartially purified proteins; and (c) implanting the product of step (b)into the body of mammal to stimulate cartilage formation at the site ofimplantation.
 14. The method of claim 13, wherein said partiallypurified cartilage-derived morphogenetic proteins are obtained fromarticular cartilage or epiphyseal cartilage.
 15. The method of claim 13,wherein the matrix of step (b) comprises a cellular material.
 16. Themethod of claim 13, wherein mixing step (b) additionally comprisesmixing of viable chondroblast or chondrocytes.
 17. The method of claim5, wherein the implanting step comprises implanting subcutaneously. 18.The method of claim 5, wherein the implanting step comprises implantingsubcutaneously.
 19. The method of claim 5, wherein the implanting stepcomprises implanting intramuscularly.
 20. A composition that can beadministered to a mammal for the purpose of stimulating chondrogenicactivity at the site of administration without substantially stimulatingosteogenic activity, said composition comprising at least onecartilage-derived morphogenetic protein and a matrix.
 21. Thecomposition of claim 20, wherein said cartilage-derived morphogeneticprotein is derived from an extract of cartilage tissue.
 22. Thecomposition of claim 20, wherein said cartilage tissue is selected fromthe group consisting of articular cartilage and epiphyseal cartilage.23. The composition of claim 20, wherein said cartilage-derivedmorphogenetic protein is a recombinant protein.
 24. The composition ofclaim 20, wherein said recombinant protein has a sequence selected fromthe group consisting of SEQ ID NO: 13 and SEQ ID NO:
 14. 25. Thecomposition of claim 20, wherein said matrix is selected from the groupconsisting of fibrin glue, freeze-dried cartilage, collagens andguanidinium-insoluble collagenous residue of demineralized bone.
 26. Thecomposition of claim 20, wherein said matrix is a non-resorbable matrixselected from the group comprising tetracalcium phosphate andhydroxyapatite.
 27. An isolated DNA molecule which codes for a proteinof the TGF-β family, wherein said DNA molecule comprises a sequenceselected from the group consisting of: (a) a part of SEQ ID NO: 11 whichencodes the mature protein, and (b) a nucleotide sequence which encodesa portion of the amino acid sequence according to SEQ ID NO: 13, whereinsaid portion is the mature protein.